Simultaneous Analysis of the p16 Gene and Protein in Canine Lymphoma Cells and Their Correlation with pRb Phosphorylation.

Cyclin-dependent kinase inhibitor p16 (CDKN2A) primarily functions as a negative regulator of the retinoblastoma protein (pRb) pathway to prevent pRb phosphorylation, thus playing a critical role in cell cycle arrest. In canine lymphoma cells, methylation due to inactivation of the p16 gene has been reported. However, its protein expression has not been examined in previous studies. In our in vitro study, the gene and protein expression of p16 and phosphorylated pRb were examined simultaneously in eight canine lymphoma and leukemia cell lines (17-71, CLBL-1, GL-1, CLC, CLGL-90, Ema, Nody-1, and UL-1). Methylation of the p16 gene was also explored using the demethylation drug 5-Aza-2'-deoxycytidine (5-Aza). After 5-Aza treatment, p16 gene and protein expression increased and pRb phosphorylation decreased, suggesting that both hypermethylation of the p16 gene and pRb hyperphosphorylation occurred in four out of eight cell lines (CLBL-1, CLC, Nody-1, and UL-1). Moreover, the estimation of p16's protein expression was better than that of p16's mRNA expression because the expression of the protein was more stable than those of the gene, and highly related to the phosphorylation of pRb. These results revealed that p16's protein expression could be a promising biomarker for canine lymphoma cells.

Unveiling the transcriptome alteration of POMC neuron in diet-induced obesity.

Loss of neuron homeostasis in the arcuate nucleus (ARC) is responsible for diet-induced-obesity (DIO). We previously reported that loss of Rb1 gene compromised the homeostasis of anorexigenic POMC neurons in ARC and induced obesity in mice. To evaluate the development of DIO, we propose to analyze the transcriptomic alteration of POMC neurons in mice following high fat diet (HFD) feeding. We isolated these neurons from established DIO mice and performed transcriptomic profiling using RNA-seq. In total, 1066 genes (628 upregulated and 438 downregulated) were identified as differentially expressed genes (DEGs). Pathway enrichment analysis with these DEGs further revealed that "cell cycle," "apoptosis," "chemokine signaling," and "sphingolipid metabolism" pathways were correlated with DIO development. Moreover, we validated that the pRb protein, a key regulator of "cell cycle pathway," was inactivated by phosphorylation in POMC neurons by HFD feeding. Importantly, the reversal of deregulated cell cycle by stereotaxic delivering of the unphosphorylated pRbΔP in ARC significantly meliorated the DIO. Collectively, our study provides insights into the mechanisms related to the loss of homeostasis of POMC neurons in DIO, and suggests pRb phosphorylation as a potential intervention target to treat DIO.

Computational approach for generating robust models for discovering novel molecules as Cyclin Dependent Kinase 4 inhibitors.

Cyclin Dependent Kinase 4 is a striking target for the proposal of anti-cancer drugs since its overexpression is associated with various types of cancers. In the present study, 2D and 3D atom based QSAR study were accomplished with 6 component PLS factor for 230 pyrido[2,3-d]pyramidine correspondents along with flexible ligand docking in the extra precision mode with the application of core constraints followed by the binding energy determinations. Kernel based partial least square analysis fitting with fingerprints initially created worthy models, among which the one with molprint2D fingerprints generated a noble model with a score value of 0.8322. Atom Based 3D QSAR resulted in an effective model with Regression coefficient (R2 = 0.8372), and Q2 = 0.7381. Docking experiments exposed hydrogen bonding interactions with hinge region residues, salt bridge formation and л-л stacking interaction as the leading non-covalent interactions causative of the inhibitory activity of CDK4 inhibitors. The primary factors that induced the stability of protein-ligand complex are the van der Waals interactions, lipophilic interactions and coulombic interactions.

Fragment-Based Discovery of 7-Azabenzimidazoles as Potent, Highly Selective, and Orally Active CDK4/6 Inhibitors.

Herein, we describe the discovery of potent and highly selective inhibitors of both CDK4 and CDK6 via structure-guided optimization of a fragment-based screening hit. CDK6 X-ray crystallography and pharmacokinetic data steered efforts in identifying compound 6, which showed >1000-fold selectivity for CDK4 over CDKs 1 and 2 in an enzymatic assay. Furthermore, 6 demonstrated in vivo inhibition of pRb-phosphorylation and oral efficacy in a Jeko-1 mouse xenograft model.